Reactions that are selective and orthogonal to (i.e., non-interacting with) the functionality found in biological systems are broadly useful tools with applications that span synthesis, chemical biology, and materials science. However, despite being grounded in some of the most venerable name reactions in organic synthesis, the development of reaction types that can selectively interface with biological molecules is a recent advance and continuing challenge. Examples of such bio-orthogonal coupling reactions include Native Chemical Ligation (NCL) and Expressed Protein Ligation (EPL), carbonyl ligations, Diels-Alder reactions, Pd— and Rh-catalyzed ligations, decarboxylative condensations, thioacid/azide ligations, aziridine ligations, the Staudinger ligation, and the Sharpless-Huisgen cycloaddition. The latter two reactions, both of which involve reactivity of azides, are shown below.

These reactions are often cited as examples of ‘click chemistry,’ a term used in the art to refer to chemical reactions that are specific, high yielding, and tolerant of functional groups. Click reactions do not generate significant byproducts (or only inoffensive byproducts), and can take place in a variety of reaction media, including water. Such reactions are orthogonal to most of the functional groups found in biological systems, and accordingly click chemistry has become a broadly useful tool for chemical biology and materials science.
The high selectivity and broad compatibility of these reactions are manifested in a variety of reaction environments, even in living cells. Furthermore, it has proven possible to express proteins that incorporate either alkynes or azides, providing a handle for protein ligation.
Despite the above advantages, these reactions are not without drawbacks. These include the cytotoxicity of the copper catalyst in the Sharpless-Huisgen reaction with simple alkynes and competing phosphine oxidation for the Staudinger reaction. Furthermore, the reaction rates are often slow, and therefore require high concentrations and/or a large excess of one reactant. This is impractical for many applications.
A further challenge for the field has been to develop new coupling reactions that are orthogonal to these existing chemistries, as well as to the functional groups typically found in biological systems. Providing such additional bio-orthogonal coupling chemistries would be very desirable as a next step in building a toolkit for preparing complex, multidomain biological structures.
The Diels-Alder reaction is one possible candidate for providing a reaction sequence that is orthogonal to the functional groups used in the above reactions as well as those typically found in biological systems. Diels-Alder reactions employing maleimide derivatives as dienophiles have been used in bioconjugation reactions of oligonucleotides, carbohydrates, and peptides. Such reactions have been used to immobilize biological molecules onto surfaces, and as a coupling tool for proteins. However, maleimide-based Diels-Alder couplings suffer from two major limitations: relatively slow reaction rates, and the incompatibility with free thiols. Thus, high concentrations (>1 mM) are typically required for the bioconjugations of diene-modified oligonucleotides (2-24 h at 37° C.). A more severe limitation is that free thiols react with maleimide via conjugate addition. As one solution, Waldmann has shown that Ellman's reagent can be used to protect the thiol groups in the surface cysteines of Rab7. However, a method that tolerates thiols would obviate the need for such a step, and would be a very desirable advance.
One possible way of addressing the problem of thiol-reactivity of the dienophile might be to use a different kind of dienophile/diene combination. One possible such combination would be an Inverse Electron Demand Diels-Alder (IED-DA) pair of reactants. For example, 1,2,4,5-tetrazine derivatives are well known as powerful dienes in inverse electron demand Diels-Alder (IED-DA) reactions of alkenes and alkynes. Such reactions of 1,2,4,5-tetrazines are typically followed by a retro-Diels-Alder reaction to expel nitrogen. Strained alkenes are particularly reactive in (IED-DA) reactions, and the kinetics of IED-DA reactions of strained molecules with 1,2,4,5-tetrazine derivatives 1a and 1b have been elucidated by J. Sauer as shown below.
All of these reactions initially produce structures 2. For certain dienophiles (e.g., trans-cyclooctene) the initially formed product 2 tautomerizes to 3. Remarkably, the rates with various dienophiles span 7 orders of magnitude. In particular, trans-cyclooctene, cyclopropene, and 3-methylcyclopropene display high reactivity.
Unfortunately, although IED-DA reactions involving 1,2,4,5-tetrazines can be very rapid, such reactions using known 1,2,4,5-tetrazines have some particularly troublesome shortcomings relative to their potential use in many applications, especially in aqueous systems such as biological media. For example, dienes 1a and 1b are themselves reactive toward water. Other tetrazines, such as 3,6-di-(2-pyridyl)-1,2,4,5-tetrazine (4), are more stable to aqueous conditions but react only slowly in water with dienophiles such as styrenes, making such reactions impractical in many applications. Further, the reactions of 4 had not been demonstrated in the presence of biological molecules or media (e.g., cellular lysate or cell culture media).

On the other hand, while cyclopropene and trans-cyclooctene have been known to display exceptional reactivity toward tetrazines in IED-DA chemistry, the synthesis of suitable derivatives has been a serious limitation. For cyclopropenes, fast reactivity has typically been found only for those compounds bearing a single substituent at C-3. For example, as shown above, the reaction of a tetrazine with 3-methylcyclopropene is ˜7000 faster than the analogous reaction with 3,3-dimethylcyclopropene. However, cyclopropenes with a single C-3 substituent have typically been too reactive to be stored, much less used in bioconjugation. For example, 3-methylcyclopropene is too reactive to be stored as a neat material, and is generally used immediately upon generation.
As seen above, trans-cyclooctene is a remarkably reactive dienophile in IED-DA reactions. However, although there are a number of preparations of the parent compound, syntheses of substituted trans-cyclooctenes are relatively few. Typically, trans-cyclooctenes are prepared via several chemical steps from the analogous cis-cyclooctene (or a more expensive/complex precursor). Far superior would be a direct synthesis of the trans- from the cis-isomer. Efforts have been made to achieve this through sensitized photolysis, but unfortunately such photochemical syntheses have typically been not preparatively useful because high dilution has been required, and because photolysis produces a mixture of cis- and trans-cyclooctene that needs to be separated by washing the mixture with aqueous AgNO3 (only the trans-isomer coordinates strongly to AgNO3, forming a water-soluble complex).
Perhaps due to the above-mentioned impediments, the inventors are unaware of any known, practically useful Diels-Alder based chemistries that are tolerant of aqueous environments, tolerant of thiol and amine groups, and of sufficient reaction rate as to have broad utility in biological systems.